1. Field of the Invention
The present invention relates to a large pore resin useful for the synthesis of high MW peptides and proteins (protides) and conjugates thereof, and to the use of the resin/high MW protide conjugate to induce an immune response in experimental animals. More particularly, the present invention relates to a novel large pore polyacrylamide resin, a method of preparing the polyacrylamide resin, a resin/high MW protide conjugate, a method of preparing a resin/high MW protide conjugate, a method of inducing immune response in a mammal with the conjugate, and a method of using the resin/high MW protide conjugate for the affinity purification of antibodies and for immunodiagnostic purposes.
2. Description of the Background
Solid phase peptide synthesis is a valuable tool for investigating the structure and mechanism of action of proteins. Most such synthetic methods involve the use of cross-linked polystyrene resins as the solid phase to which the peptide is anchored during assembly, usually through a linker molecule. Assembly is accomplished by a repetitive cycle of adding a protected amino acid to the solid phase, selectively removing a protective group on that amino acid (deprotecting) and adding the next protected amino acid.
Although cross-linked, polystyrene resins have been used as supports in solid phase peptide synthesis, their relatively hydrophobic character in comparison to the polar organic media required to solubilize reactants can be problematic in peptide chain assembly. Such media may freely solvate a growing peptide, yet incompletely swell the polystyrene matrix. Within the polymer lattice, impaired diffusion of reagents and steric hindrance can contribute to lowered efficiency during coupling cycles, which, on a repeated basis, lowers final yields appreciably. During the early stages of assembly, when the resin to peptide mass ratio is high and the physical properties of the support dominate, this lowered efficiency is particularly acute.
Those shortcomings led to the development of a cross-linked, polydimethylacrylamide support which is highly polar in character and freely permeated by the requisite solvents for peptide synthesis. Such polyamide resin, as the aminomethyl derivative, permits the use of synthetic schemes incorporating alternate protection strategies through selection of appropriate linker molecules, which link C-terminal residues to the support. The thus synthesized peptide or protein, referred to herein as a "protide", may be used in a number of applications.
Of particular interest to the present invention is the use of a protide as an immunogen. It has previously been demonstrated that synthetic peptides analogous to sequences contained in vital-encoded proteins have proven useful for identifying native antigen determinants associated with such proteins, such as various HBsAg synthetic peptides. The induction of an antibody response to HBsAg, using such peptides, was proven to be relatively weak. However, this response could be enhanced through coupling of peptides to a carrier protein prior to immunization.
Moreover, the prediction of potential antigenic determinants of immunogenic proteins based on primary sequences analysis is far from being exact. Because of this, the identification of putative epitopes, through trial and error, may be laborious.
Affinity adsorbents have been prepared using commercially-available activated gels such as Bio-Gel P and Bio-Gel A (Bio-Rad), as well as conjugates of Sepharose or Agarose (Pharmacia) with low molecular weight substances. Polydimethylacrylamide resins have also been utilized as conjugates with small peptides for the purification of antibodies.
In addition, small peptide/polyamide resin conjugates were synthesized and utilized for the immunization of experimental animals. Previous technology permitted the synthesis on a solid polyamide resin support of amino acid sequences of limited length. In general, amino acid sequences of only up to twenty-five or thirty amino acids can be attained by previous methods.
There is still a need, therefore, for a method of preparing large synthetic peptides or proteins mimmicking native antigenic sequences which neither requires the purification of the synthetic peptide nor its coupling to a carrier protein.